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1.  Developing a denoising filter for electron microscopy and tomography data in the cloud 
Biophysical reviews  2012;4(3):223-229.
The low radiation conditions and the predominantly phase-object image formation of cryo-electron microscopy (cryo-EM) result in extremely high noise levels and low contrast in the recorded micrographs. The process of single particle or tomographic 3D reconstruction does not completely eliminate this noise and is even capable of introducing new sources of noise during alignment or when correcting for instrument parameters. The recently developed Digital Paths Supervised Variance (DPSV) denoising filter uses local variance information to control regional noise in a robust and adaptive manner. The performance of the DPSV filter was evaluated in this review qualitatively and quantitatively using simulated and experimental data from cryo-EM and tomography in two and three dimensions. We also assessed the benefit of filtering experimental reconstructions for visualization purposes and for enhancing the accuracy of feature detection. The DPSV filter eliminates high-frequency noise artifacts (density gaps), which would normally preclude the accurate segmentation of tomography reconstructions or the detection of alpha-helices in single-particle reconstructions. This collaborative software development project was carried out entirely by virtual interactions among the authors using publicly available development and file sharing tools.
PMCID: PMC3467108  PMID: 23066432
cryo-EM; Digital paths; Filtering; Helix detection; Supervised classification; Remote collaboration
2.  Computational opportunities for remote collaboration and capacity building afforded by Web 2.0 and cloud computing 
Biophysical reviews  2012;4(3):153-160.
In this paper, we state our aims and aspirations for building a global network of likeminded people interested in developing and encouraging students in the field of computational biophysics (CB). Global capacity building efforts have uncovered local computational talent in virtually every community regardless of where the students reside. Our vision is to discover and encourage these aspiring investigators by suggesting ways that they and other “garage scientists” can participate in new science even if they have no access to sophisticated research infrastructure. We argue that participatory computing in the “cloud” is particularly suitable for CB and available to any budding computational biophysicist if he or she is provided with open-minded mentors who have the necessary skills and generosity. We recognize that there are barriers to the development of such remote collaborations, and we discuss possible pathways to overcome these barriers. We point out that this Special Issue of Biophysical Reviews provides a much-needed forum for the development of several specific applications of CB.
PMCID: PMC3467109  PMID: 23066431
Computational biophysics; White paper; Cloud computing; Capacity building; Garage science; Global network
3.  Automated Tracing of Filaments in 3D Electron Tomography Reconstructions using Sculptor and Situs 
Journal of structural biology  2012;178(2):121-128.
The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7 and Sculptor 2.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.
PMCID: PMC3440181  PMID: 22433493
Tomograms; 3D analysis; filament detection; actin networks; denoising; segmentation
4.  Evolutionary Bidirectional Expansion for the Tracing of Alpha Helices in Cryo-Electron Microscopy Reconstructions 
Journal of Structural Biology  2011;177(2):410-419.
Cryo-electron microscopy (cryo-EM) enables the imaging of macromolecular complexes in near-native environments at resolutions that often permit the visualization of secondary structure elements. For example, alpha helices frequently show consistent patterns in volumetric maps, exhibiting rod-like structures of high density. Here, we introduce VolTrac (Volume Tracer) - a novel technique for the annotation of alpha-helical density in cryo-EM data sets. VolTrac combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to trace helical regions. Our method takes advantage of the stochastic search by using a genetic algorithm to identify optimal placements for a short cylindrical template, avoiding exploration of already characterized tabu regions. These placements are then utilized as starting positions for the adaptive bidirectional expansion that characterizes the curvature and length of the helical region. The method reliably predicted helices with seven or more residues in experimental and simulated maps at intermediate (4–10 Å) resolution. The observed success rates, ranging from 70.6% to 100%, depended on the map resolution and validation parameters. For successful predictions, the helical axes were located within 2Å from known helical axes of atomic structures.
PMCID: PMC3288247  PMID: 22155667
cryo-electron microscopy; intermediate resolution; extract alpha helices; annotate alpha helices; secondary structure elements
5.  Using Sculptor and Situs for Simultaneous Assembly of Atomic Components into Low-Resolution Shapes 
Journal of Structural Biology  2010;173(3):428-435.
We describe an integrated software system called Sculptor that combines visualization capabilities with molecular modeling algorithms for the analysis of multi-scale data sets. Sculptor features extensive special purpose visualization techniques that are based on modern GPU programming and are capable of representing complex molecular assemblies in real-time. The integration of graphics and modeling offers several advantages. The user interface not only eases the usually steep learning curve of pure algorithmic techniques, but it also permits instant analysis and post-processing of results, as well as the integration of results from external software. Here, we implemented an interactive peak-selection strategy that enables the user to explore a preliminary score landscape generated by the colores tool of Situs. The interactive placement of components, one at a time, is advantageous for low-resolution or ambiguously shaped maps, which are sometimes difficult to interpret by the fully automatic peak selection of colores. For the subsequent refinement of the preliminary models resulting from both interactive and automatic peak selection, we have implemented a novel simultaneous multi-body docking in Sculptor and Situs that softly enforces shape complementarities between components using the normalization of the cross-correlation coefficient. The proposed techniques are freely available in Situs version 2.6 and Sculptor version 2.0.
PMCID: PMC3352678  PMID: 21078392
multi-resolution modeling; multi-component registration; macromolecular assembly; cryo-electron microscopy; immersive visualization; haptic rendering
6.  Polymorphism of the Epidermal Growth Factor Receptor Extracellular Ligand Binding Domain: The Dimer Interface Depends on Domain Stabilization 
Biochemistry  2011;50(12):2144-2156.
Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play important roles in cell proliferation and signaling. EGFR extracellular domain (sEGFR) forms a dimer upon the binding of ligands, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα). In this study, multiple molecular dynamics (MD) simulations of the 2:2 EGF:sEGFR3–512 dimer and the 2:2 TGFα:sEGFR3–512 dimer were performed in solvent and crystal environments. The simulations of systems comprising up to half a million atoms reveal part of the structural dynamics that sEGFR dimers are capable of. The solvent simulations consistently exhibited a prominent conformational relaxation from the initial crystal structures on the nanosecond time scale, leading to a symmetry breaking and more extensive contacts between the two sEGFR monomers. In the crystal control simulation, this symmetry breaking and compaction was largely suppressed by crystal packing contacts. The simulations also provided evidence that the disordered domain IV of sEGFR may act as a stabilizing spacer in the dimer. Thus the simulations suggest that the sEGFR dimer can take diverse configurations in solvent environments. These biologically relevant conformations of the EGFR signal transduction network can be controlled by contacts among the structural domains of sEGFR and its ligands.
PMCID: PMC3062653  PMID: 21275429
epidermal growth factor; transforming growth factor α; receptor; dimer; crystal packing; buttressing; spacer
7.  Conventions and workflows for using Situs  
Recent developments of the Situs software suite for multi-scale modeling are reviewed. Typical workflows and conventions encountered during processing of biophysical data from electron microscopy, tomography or small-angle X-ray scattering are described.
Situs is a modular program package for the multi-scale modeling of atomic resolution structures and low-resolution biophysical data from electron microscopy, tomography or small-angle X-ray scattering. This article provides an overview of recent developments in the Situs package, with an emphasis on workflows and conventions that are important for practical applications. The modular design of the programs facilitates scripting in the bash shell that allows specific programs to be combined in creative ways that go beyond the original intent of the developers. Several scripting-enabled functionalities, such as flexible transformations of data type, the use of symmetry constraints or the creation of two-dimensional projection images, are described. The processing of low-resolution biophysical maps in such workflows follows not only first principles but often relies on implicit conventions. Situs conventions related to map formats, resolution, correlation functions and feature detection are reviewed and summarized. The compatibility of the Situs workflow with CCP4 conventions and programs is discussed.
PMCID: PMC3322594  PMID: 22505255
Situs; multi-resolution data
8.  Matching Structural Densities from Different Biophysical Origins with Gain and Bias 
Journal of structural biology  2010;173(3):445-450.
The registration of volumetric structures in real space involves geometric and density transformations that align a target map and a probe map in the best way possible. Many computational docking strategies exist for finding the geometric transformations that superimpose maps, but the problem of finding an optimal density transformation, for the purposes of difference calculations or segmentation, has received little attention in the literature. We report results based on simulated and experimental electron microscopy maps, showing that a single scale factor (gain) may be insufficient when it comes to minimizing the density discrepancy between an aligned target and probe. We propose an affine transformation, with gain and bias, that is parameterized by known surface isovalues and by an interactive centering of the “cancellation peak” in the surface thresholded difference map histogram. The proposed approach minimizes discrepancies across a wide range of interior densities. Owing to having only two parameters, it avoids overfitting and requires only minimal knowledge of the probe and target maps. The linear transformation also preserves phases and relative amplitudes in Fourier space. The histogram matching strategy was implemented in the newly revised volhist tool of the Situs package, version 2.6.
PMCID: PMC3246903  PMID: 20854911
Difference mapping; discrepancy; volumetric maps; docking; electron microscopy; thick filament
9.  Outcome of the First Electron Microscopy Validation Task Force Meeting 
Structure(London, England:1993)  2012;20-330(2):205-214.
This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM ( and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.
PMCID: PMC3328769  PMID: 22325770
10.  Using Situs for the integration of multi-resolution structures 
Biophysical Reviews  2010;2(1):21-27.
Situs is a modular and widely used software package for the integration of biophysical data across the spatial resolution scales. It has been developed over the last decade with a focus on bridging the resolution gap between atomic structures, coarse-grained models, and volumetric data from low-resolution biophysical origins, such as electron microscopy, tomography, or small-angle scattering. Structural models can be created and refined with various flexible and rigid body docking strategies. The software consists of multiple, stand-alone programs for the format conversion, analysis, visualization, manipulation, and assembly of 3D data sets. The programs have been ported to numerous platforms in both serial and shared memory parallel architectures and can be combined in various ways for specific modeling applications. The modular design facilitates the updating of individual programs and the development of novel application workflows. This review provides an overview of the Situs package as it exists today with an emphasis on functionality and workflows supported by version 2.5.
Electronic supplementary material
The online version of this article (doi:10.1007/s12551-009-0026-3) contains supplementary material, which is available to authorized users.
PMCID: PMC2821521  PMID: 20174447
Structural models; 3D data sets; Multi-platform; Modeling
11.  Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity 
Journal of molecular biology  2008;384(4):780-797.
Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free-head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens reveals that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal here has been to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction reveals intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC, and fitted to the reconstruction an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 crystal structure. The fitting suggests an intramolecular interaction between the cardiomyopathy loop of the free-head and its own S2 and two intermolecular interactions—between the cardio-loop of the free head and the ELC of the blocked head, and between the Leu-305 - Gln-327 “interaction loop” (loop I) of the free-head and the N-terminal fragment of the RLC of the blocked-head. These interactions, added to those previously described, would help to switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.
PMCID: PMC2729561  PMID: 18951904
Myosin regulation; thick filament; cryo-EM; myosin regulatory light chain; phosphorylation
12.  Coarse-Graining Protein Structures With Local Multivariate Features from Molecular Dynamics 
The Journal of Physical Chemistry. B  2008;112(44):14026-14035.
A multivariate statistical theory, local feature analysis (LFA), extracts functionally relevant domains from molecular dynamics (MD) trajectories. The LFA representations, like those of principal component analysis (PCA), are low dimensional and provide a reduced basis set for collective motions of simulated proteins, but the local features are sparsely distributed and spatially localized, in contrast to global PCA modes. One key problem in the assignment of local features is the coarse-graining of redundant LFA output functions by means of seed atoms. One can solve the combinatorial problem by adding seed atoms one after another to a growing set, minimizing a reconstruction error at each addition. This allows for an efficient implementation, but the sequential algorithm does not guarantee the optimal mutual correlation of the sequentially assigned features. Here, we present a novel coarse-graining algorithm for proteins that directly minimizes the mutual correlation of seed atoms by Monte Carlo (MC) simulations. Tests on MD trajectories of two biological systems, bacteriophage T4 lysozyme and myosin II motor domain S1, demonstrate that the new algorithm provides statistically reproducible results and describes functionally relevant dynamics. The well-known undersampling of large-scale motion by short MD simulations is apparent also in our model, but the new coarse-graining offers a major advantage over PCA; converged features are invariant across multiple windows of the trajectory, dividing the protein into converged regions and a smaller number of localized, undersampled regions. In addition to its use in structure classification, the proposed coarse-graining thus provides a localized measure of MD sampling efficiency.
PMCID: PMC2651751  PMID: 18855436
13.  Biomolecular pleiomorphism probed by spatial interpolation of coarse models 
Bioinformatics  2008;24(21):2460-2466.
In low resolution structures of biological assemblies one can often observe conformational deviations that require a flexible rearrangement of structural domains fitted at the atomic level. We are evaluating interpolation methods for the flexible alignment of atomic models based on coarse models. Spatial interpolation is well established in image-processing and visualization to describe the overall deformation or warping of an object or an image. Combined with a coarse representation of the biological system by feature vectors, such methods can provide a flexible approximation of the molecular structure. We have compared three well-known interpolation techniques and evaluated the results by comparing them with constrained molecular dynamics. One method, inverse distance weighting interpolation, consistently produced models that were nearly indistinguishable on the alpha carbon level from the molecular dynamics results. The method is simple to apply and enables flexing of structures by non-expert modelers. This is useful for the basic interpretation of volumetric data in biological applications such as electron microscopy. The method can be used as a general interpretation tool for sparsely sampled motions derived from coarse models.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2732278  PMID: 18757874
14.  Actin Depolymerizing Factor Stabilizes an Existing State of F-Actin and Can Change the Tilt of F-Actin Subunits 
The Journal of Cell Biology  2001;153(1):75-86.
Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large (∼12°) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.
PMCID: PMC2185532  PMID: 11285275
actin; ADF; cooperativity; electron microscopy; image processing

Results 1-14 (14)